Diagnostic method for cobalamin deficiency

Abstract

A method of diagnosing cobalamin deficiency in warm-blooded animals is described. In one embodiment, this method comprises the measurement of methylmalonic acid levels in cerebral spinal fluid to determine if such levels exceed normal range. A finding of elevated levels indicates a cobalamin deficiency. In another embodiment, the method comprises the measurement of methylmalonic acid levels in cerebral spinal fluid and serum. A finding that the ratio of methylmalonic acid in cerebral spinal fluid to that in serum is above normal is indicative of a cobalamin deficiency. Additionally, a method for assessing the effectiveness of cobalamin therapy is described. The effectiveness of therapy is assessed by determining whether cerebral spinal fluid levels of methylmalonic acid have returned to normal, or by determining whether the ratio of methylmalonic acid in cerebral spinal fluid to that in serum has returned to normal range. A method for distinguishing cobalamin deficiency from renal failure in warm-blooded animals having excessive serum MMA levels is also described. This method comprises determining the ratio of CSF MMA to serum MMA. A finding that the CSF MMA/serum MMA ratio is above normal or elevated is indicative of the presence of Cbl deficiency, whereas a finding that the CSF MMA/serum MMA ratio is below normal is indicative of renal failure.

We claim: 
     
       1. A method for detecting cobalamin deficiency in a human by assaying the levels of methylmalonic acid in the cerebral spinal fluid and serum of said human to determine the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio exceeds a predetermined range, wherein an excessive ratio is indicative of cobalamin deficiency, and wherein said method comprises the steps of: (a) adding a known amount of an internal standard to said cerebral spinal fluid, said internal standard comprising methylmalonic acid labeled with a stable isotope marker,   (b) adjusting the pH of said cerebral spinal fluid to about 1,   (c) extracting said acidified fluid with ether,   (d) applying said ether extract to a silica gel column which has been washed and equilibrated with ether,   (e) washing said applied extract on said column with methanol,   (f) eluting a fraction containing methylmalonic acid and internal standard by applying a mixture of about 4N NH 4  OH in methanol to said column containing said applied extract,   (g) collecting said eluted fraction and derivatizing methylmalonic acid and internal standard contained therein,   (h) determining methylmalonic acid concentration in said cerebral spinal fluid by gas chromatography-mass spectrometry quantitation of methylmalonic acid and internal standard in said derivatized eluted fraction,   (i) repeating steps (a) through (h) using said serum of said human to determine methylmalonic acid concentration in said serum, and   (j) determining the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio exceeds normal range, wherein excessive ratio is indicative of cobalamin deficiency.   
     
     
       2. A method of assessing the adequacy of cobalamin therapy in a human having cobalamin deficiency which comprises assaying cerebral spinal fluid and serum of said human according to the method of claim 1 after administration of said cobalamin therapy, to determine the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio has returned to a predetermined range, wherein return to said predetermined range is indicative of the adequacy of the therapy. 
     
     
       3. An improved method for treatment of cobalamin deficiency by administration of exogenous cobalamin in humans wherein the improvement comprises the assessment of the adequacy of cobalamin therapy by assaying cerebral spinal fluid and serum according to the method of claim 1 after administration of said exogenous cobalamin to determine the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio has returned to a predetermined range, wherein return to said predetermined range is indicative of adequacy of said therapy. 
     
     
       4. A method for detecting cobalamin deficiency in a human by assaying the levels of methylmalonic acid in the cerebral spinal fluid and serum of said human to determine the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio exceeds a predetermined range, wherein an excessive ratio is indicative of cobalamin deficiency, and wherein said method comprises the steps of: (a) adding a known amount of an internal standard to said cerebral fluid, said internal standard comprising methylmalonic acid labeled with a stable isotope marker,   (b) adjusting the pH of said fluid of step (a) to about 12,   (c) extracting said basic cerebral spinal fluid of step (b) with ether and discarding said ether extract,   (d) adjusting the pH of said extracted cerebral spinal fluid of step (c) to about 1,   (e) extracting the acidic cerebral spinal fluid of step (d) with ether and collecting said ether extract,   (f) injecting said extract of step (e) onto a high performance liquid chromatography anion exchange system having a mobile phase consisting of about 0.05M KH 2  PO 4  --H 3  PO 4  at pH 2,   (g) collecting a fraction containing methylmalonic acid and internal standard,   (h) adjusting the pH of said fraction of step (g) to about 1,   (i) extracting said acidified fraction of step (h) with ether,   (j) derivatizing methylmalonic acid and internal standard in said ether fraction of step (i),   (k) determining methylmalonic acid concentration in said cerebral spinal fluid by quantitating derivatized methylmalonic acid and internal standard in said derivatized fraction of step (j) by gas chromatograph-mass spectrometry,   (l) repeating steps (a) through (k) using said serum of said human to determine methylmalonic acid concentration in said serum, and   (m) determining the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio exceeds normal range, wherein excessive ratio is indicative of cobalamin deficiency.   
     
     
       5. A method of assessing the adequacy of cobalamin therapy in a human having cobalamin deficiency which comprises assaying cerebral spinal fluid and serum of said human according to the method of claim 4 after administration of said cobalamin therapy, to determine the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio has returned to a predetermined range, wherein return to said predetermined range is indicative of the adequacy of the therapy. 
     
     
       6. An improved method for treatment of cobalamin deficiency by administration of exogenous cobalamin in humans wherein the improvement comprises the assessment of the adequacy of cobalamin therapy by assaying cerebral spinal fluid and serum according to the method of claim 4 after administration of said exogenous cobalamin to determine the ratio of methylmalonic acid in cerebral spinal fluid to that in serum, to assess whether said ratio has returned to a predetermined range, wherein return to said predetermined range is indicative of adequacy of said therapy.